Experiences with Ingelvac MycoFLEX Vaccine and FLEXcombo in Australia

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1 Experiences with Ingelvac MycoFLEX Vaccine and FLEXcombo in Australia Ingelvac MycoFLEX vaccine was launched into Australia late in 2015 to protect pigs from Mycoplasma hyopneumoniae (M. hyo). The vaccine also has the added benefit of being able to be mixed with Ingelvac CircoFLEX vaccine to provide protection against Porcine Circovirus Associated Disease (PCVAD) as well in just one injection. The combination of the two products is referred to as FLEXcombo. To assist with the fresh mixing of Ingelvac CircoFLEX vaccine with Ingelvac MycoFLEX vaccine Boehringer Ingelheim have developed the Head Space Bottle (HSB) for Ingelvac MycoFLEX vaccine. The extra head space in the bottle allows the correct volume of Ingelvac CircoFLEX vaccine to be mixed quickly and easily using a specially designed transfer needle. Vaccination equipment can then be attached directly to the bottle for protection against both M. hyo and PCVAD in one injection. The studies attached are three examples of where Ingelvac MycoFLEX vaccine and FLEXcombo have been used with success on commercial piggeries in Australia. These studies also incorporated the Boehringer Ingelheim Cough Control Program for the monitoring of clinical respiratory disease on farm as well as growth rates and lung lesion scores post slaughter also affected by respiratory disease. Boehringer Ingelheim would like to thank the farmers and veterinarians involved in the running of these studies. Study 1 A co-mingled field trial on a commercial Australian farm comparing the efficacy of FLEXcombo with a two shot M. hyo vaccination program. Method This field trial was conducted to compare the efficacy of FLEXcombo with the farm s current vaccination program using two separate M. hyo vaccinations. The trial was conducted over six months through the summer and autumn (December-May) with 2,014 mixed sex piglets enrolled over two weeks of production. Piglets were followed through until slaughter at 20 weeks of age. Piglets were allocated to treatment groups by alternate selection at processing (one day of age) as they presented to farm workers in the farrowing crate. At processing all piglets were allocated, tagged, weighed and tailed. The tag was made up of an individual electronic identification tag and a colored tag indicating treatment group. The piglets from Group 1 were vaccinated with 2 ml of a competitor M. hyo vaccine registered for use without a booster dose. Piglets were allocated to make up an individual batch for each week from several different farrowing houses on the property.

2 Treatment Group 1 Number Treatment Age (days) Tag Color Competitor one shot M. hyo 1 vaccine (2 ml) 1,010 Ingelvac CircoFLEX (1 ml) Orange Treatment Group 2 Competitor two shot M. hyo vaccine (2 ml) 56 (approx.) Number Treatment Age (days) Tag Color 1,004 FLEXcombo (2 ml) Green At days of age piglets from Treatment Group 1 were vaccinated with 1 ml of Ingelvac CircoFLEX (Boehringer Ingelheim Pty Limited). Piglets in Treatment Group 2 were given a 2 ml dose of FLEXcombo (Boehringer Ingelheim Pty Limited) a combination of 1 ml of Ingelvac MycoFLEX (Boehringer Ingelheim Pty Limited) and 1 ml of Ingelvac CircoFLEX freshly mixed within four hours of injection. Piglets remained in their farrowing sheds till weaning at days of age and were then moved to a single nursery facility. Each batch was kept predominantly separate with approximately 15% of piglets being mixed between both weeks dependent on space requirements in the nursery and grower facilities. Both treatment groups were co-mingled right through the study. Treatment Group 1 was vaccinated with 2 ml of a two shot competitor M. hyo vaccine as a booster at approximately 56 days of age. This was completed by the normal practice of the farm by vaccinating the pigs in their nursery pen. To ensure that Treatment Group 2 were not vaccinated they were marked with green stock marker before Treatment Group 1 pigs were vaccinated. Respiratory disease was monitored clinically via cough scores or Cough Indexes (CI) through the study using the Boehringer Ingelheim Cough Control Program (BICCP). This included a baseline indication of respiratory disease taken before the start of the trial. The BICCP was modelled on the diagnosis of enzootic pneumonia as described by Nathues 1. Pigs were individually weighed at approximately 20 weeks of age before any pigs were selected for slaughter. Data was analyzed using a two tailed t-test in Statistica (Version 9).

3 Results Mortality Up to Day 21 Post Day 21 Competitor Program 13.2% 6.9% FLEXcombo 13.6% 4.8% Cause of death was diagnosed and recorded by farm staff with guidance from the consulting veterinarian. Reasons for death included layovers, sudden deaths, destruction, lameness, meningitis and Actinobacillus pleuropneumonia (APP). Boehringer Ingelheim Cough Control Program Age (weeks) Baseline CI* (%) October 2015 Trial Pigs CI* (%) February, March, April *Cough Index <2.5% indicates M. hyo infection is under control. Cough Indexes were predominantly reduced in the trial pigs compared with the baseline data. The Cough Indexes indicated that respiratory disease according to M. hyo was under control 1. Live Weights FLEXcombo Competitor Vaccine Program Difference p value 20 week Live Weight (kg) ADG (kg) There was no statistically significant difference between the treatment groups for live weights or growth rates taken at 20 weeks of age. Conclusion In this example on a commercial farm in Australia, FLEXcombo provided one shot protection against M. hyo when compared with a program using multiple M. hyo vaccinations and provided significant benefits in vaccine usage and labor savings to the farmer.

4 Study 2 A co-mingled field trial on a commercial Australian, continuous flow farm comparing the efficacy of FLEXcombo with and without a booster with a two shot M. hyo vaccination program. Method This field trial was conducted to compare the efficacy of FLEXcombo with and without a booster with the farm s current vaccination program using two separate M. hyo vaccinations. The trial was conducted on a 1,500 farrow to finish property in Queensland. It ran over six months (December May) with 1,218 mixed sex piglets enrolled over two weeks of production. Piglets were followed through until slaughter from 20 weeks of age. All piglets were allocated, tagged and weighed after weaning and transported to the nursery (18-21 days of age). The nursery was attached to the farrowing shed. Weaning occurred on the Monday and Thursday of each week. Piglets were allocated to treatment groups by alternate selection. The tag was made up of an individual electronic identification tag and a colored tag indicating the treatment group. The piglets from Treatment Group 1 were vaccinated with 1 ml of M+PAC (Intervet Australia Pty Limited) and 1 ml of Ingelvac CircoFLEX (Boehringer Ingelheim Australia Pty Ltd). Piglets from Treatment Group 2 and 3 were vaccinated with 2 ml of FLEXcombo (Boehringer Ingelheim Australia Pty Ltd) a combination of 1 ml Ingelvac MycoFLEX (Boehringer Ingelheim Australia Pty Ltd) and 1 ml of Ingelvac CircoFLEX freshly mixed within four hours of injection. Treatment Group 1 Number Treatment Age (days) Tag Color M+PAC (1 ml) Ingelvac CircoFLEX (1 ml) Green M+PAC (1 ml) Treatment Group 2 Number Treatment Age (days) Tag Color 408 FLEXcombo (2 ml) M+PAC (1 ml) Purple Treatment Group 3 Number Treatment Age (days) Tag Color 403 FLEXcombo (2 ml) Orange As the system was continuous flow the first piglets enrolled in the study were mixed with piglets from the week before in their nursery pen of approximately 540 piglets. The same happened with the piglets enrolled on the Thursday of the second week also being mixed with younger piglets from the following Monday not in the study. The net result was that trial pigs were spread across three batches of piglets in the nursery. Piglets are transferred to the grower facility at approximately 60 days of age. Both treatment groups were co-mingled throughout the study.

5 Treatment Group 1 and Treatment Group 2 were vaccinated with 1 ml of M+PAC as a booster at approximately 49 days of age. Treatment Group 3 did not receive a booster M. hyo vaccination. This was completed by the farm s normal practice in the piglet s nursery pen and with piglets being marked with stock marker after vaccination for identification. Respiratory disease was monitored clinically via Cough Indexes through the study using the Boehringer Ingelheim Cough Control Program (BICCP). This was compared with a baseline indication of respiratory disease taken before the start of the trial. Blood samples were also taken for detection of M. hyo antibodies. The BICCP was modelled on the diagnosis of enzootic pneumonia as described by Nathues 1. Pigs were individually weighed at approximately 20 weeks of age as they approached slaughter weight before any trial pigs were selected for slaughter. Pigs were tattooed by treatment group at weighing so that slaughter weights could be recorded. A sample of pigs was taken at slaughter for inspection of lung pathology and lung lesion scoring. The observer was blinded and applied the lung lesion scoring system described by Muirhead and Alexander 2. Data was analyzed using a two tailed t-test in Statistica (Version 9). Results Mortality Twenty six pigs were recorded as having died in total, nine from the Treatment Group 1, five from Treatment Group 2 and twelve from Treatment Group 3. Of all these deaths only one pig was recorded as having died of respiratory disease, and this was recorded as having pneumonia. Boehringer Ingelheim Cough Control Program Age (weeks) Baseline CI* (%) October 2015 Trial Pigs CI* (%) February, March, April *Cough Index <2.5% indicates M. hyo disease under control. Cough Indexes were comparable between the trial pigs and the baseline data. The cough scores indicated that respiratory disease attributed to M. hyo was under control 1. Serology results indicate that piglets were being infected with M. hyo at approximately weeks of age.

6 Figure 1: Baseline Coughing Index and M. hyo serology result Figure 2: Treatment Groups Coughing Index and FLEXcombo M. hyo serology result

7 Average Daily Gain (ADG) ADG Treatment Group Treatment Group Treatment Group Treatment Group Treatment Group Treatment Group p value ,033 pigs were weighed before slaughter. There was no statistically significant difference in ADG between the treatment groups. Slaughter Weights Weight Treatment Group Treatment Group Treatment Group Treatment Group Treatment Group Treatment Group p value Slaughter weights were collected from 944 pigs. There were no statistical differences in slaughter weights between the groups. Lung Lesion Scores Lung score Treatment Group Treatment Group Treatment Group Treatment Group Treatment Group Treatment Group p value A sample of 201 pigs had their lungs inspected at slaughter. There was no statistical difference in lung lesions score between the treatment groups. These lung lesion scores indicate that there was no effect on ADG due to Enzootic Pneumonia 2.

8 Conclusion Under the conditions of this study on a commercial farm in Australia, FLEXcombo provided one shot protection against M. hyo when compared with a program using multiple M. hyo vaccinations and provided significant benefits in vaccine usage and labor savings to the farmer. There was no benefit for the parameters investigated in adding a booster vaccination to the one dose FLEXcombo vaccine. Study 3 A field experience on a commercial Australian, batch farrow farm comparing the efficacy of Ingelvac MycoFLEX with a single shot M. hyo vaccination. Method This field trial was conducted to compare the efficacy of Ingelvac MycoFLEX (Boehringer Ingelheim Australia Pty Ltd) with the farms previous M. hyo vaccination program using Respisure One (Zoetis Australia Pty Ltd). The trial was conducted on a 500 sow all in all out, farrow to finish property in South Australia with weekly batching. Two cohorts of 12 weeks production (4,589 piglets in total) were followed through until slaughter from 20 weeks of age. Piglets were vaccinated at approximately 19 days of age on a Monday. The first 12 weeks of production (2,233 piglets) were vaccinated with 2 ml of Respisure One. The second 12 weeks of production (2,356 piglets) were vaccinated with 1 ml of Ingelvac MycoFLEX. Both groups were given 1 ml of Ingelvac CircoFLEX (Boehringer Ingelheim Australia Pty Ltd) at the same time as the M. hyo vaccination. Original Vaccine Group Number Number of weeks production Treatment Age (days) 2, Respisure One (2 ml) 19 Ingelvac MycoFLEX Group Number Number of weeks production Treatment Age (days) 2, Ingelvac MycoFLEX (1 ml) 19 The Thursday after vaccination the piglets were weaned and transferred to a grower facility and housed in straw based shelters until 11 weeks of age. Pigs are then moved to grower shed with solid/slatted flooring. Respiratory disease was monitored clinically via the Boehringer Ingelheim Cough Control Program (BICCP). This compared the respiratory disease picture between piglets vaccinated with the two different vaccines. Blood samples were also taken for detection of M. hyo antibodies. The BICCP was modelled on the diagnosis of enzootic pneumonia as described by Nathues 1. All pigs were individually weighed at 19 weeks of age.

9 A sample of the Ingelvac MycoFLEX vaccinated pigs was taken at slaughter for inspection of lung pathology and lung lesion scoring. The observer applied the lung lesion scoring system described by Muirhead and Alexander 2. Data was analyzed using a two tailed t-test in Statistica (Version 9). Results Growth Rates Table 1: Live weight and growth rate parameters between treatments MycoFLEX n=2356 Respisure One n=2233 Difference P value End Live Weight (kg) kg <0.001 Average Daily Gain (g/day) <0.001 Pigs vaccinated with Ingelvac MycoFLEX had significantly higher live weights and ADG than pigs vaccinated with Respisure One.

10 Boehringer Ingelheim Cough Control Program Table 2: Coughing Index (CI) monitoring Age (weeks) Nov-15 CI (%) Respisure One Dec-15 CI (%) Respisure One Apr-16 CI (%) MycoFLEX May-16 CI (%) MycoFLEX Jun-16 CI (%) MycoFLEX *Cough Index (CI) <2.5% indicates M. hyo disease under control. Figure 1: Respisure One Coughing Index and M. hyo serology result

11 Figure 2: Ingelvac MycoFLEX Coughing Index and M. hyo serology result Cough Indexes were comparable between the trial pigs and the baseline data. The cough scores indicated that respiratory disease due to M. hyo was under control 1. Serology results indicate that piglets were being infected with M. hyo from 8 weeks of age onwards. The high incidence of coughing at 6-8 weeks of age in the pigs vaccinated with MycoFLEX was investigated with clinical inspection and post mortem of an affected piglet. The serum profile and post mortem investigation indicated that the coughing was unlikely to be associated with M. hyo. This has been an ongoing problem on the farm and further investigation into the cause of this coughing is warranted. The graph shows that only 20% of the serum samples collected from 6 and 8 week old pigs were positive for M. hyo antibodies. Noticeably, one sample turned out as indeterminate at 4 weeks old. This is most likely a remnant of M. hyo maternal antibodies.

12 Percentage Pigs Lung Lesion Scores Table 3: Lung Lesion Score Assessments MycoFLEX Date Number Highest Score Lowest Score Average 22 April June Average Lung Lesion Score 6.95 Figure 3: MycoFLEX lung lesion scores Lung Lesion Scores MycoFLEX Lung Lesion Score April June A total of 159 pigs had their respiratory tract inspected at slaughter across two batches of production. Lung scores ranged from 0 to 30 with an average lung lesion score of These lung lesion scores indicate that there was no effect on ADG due to Enzootic Pneumonia 2. Conclusion In this field experience on a commercial farm in Australia, Ingelvac MycoFLEX provided one shot protection against M. hyo. 1 Value of the clinical examination in diagnosing enzootic pneumonia in fattening pigs Nathues H, Spergser J, Rosengarten R, Kreienbrock L, grosse Beilage E; p443-7 The Veterinary Journal Managing Pig Health: A Reference for the Farm 2 nd Edition Muirhead MR, Alexander TJL, Carr J; p362